A study on the neuroprotective mechanism of nobiletin through regulation of mitochondrial membrane potential in primary cortical neurons and isolated brain mitochondria

Abstract

Mitochondrial calcium overload is an important event at the cellular level in determining the fate of neuronal survival and death and is regulated by a variety of channels, antiporters and pumps, as well as an electron transport chains (ETC) in the mitochondrial inner membrane. There are two motivating forces for calcium influx into the mitochondria, one of which is the calcium ion (Ca2+) concentration gradient, and the other is mitochondrial membrane potential (ΔΨm). ΔΨm is regulated within the range of -120 to -180 mV under physiological conditions. The negative charge of ΔΨm has a significant effect on Ca2+ influx. Therefore, pharmacological manipulation of ΔΨm could be a promising strategy to prevent neuronal cell death against brain insults by reducing calcium influx into the mitochondria. Based on this hypothesis, it was investigated here whether nobiletin, a Citrus polymethoxylated flavone (CPE), prevents neuronal cell death by reducing mitochondrial calcium overload and oxidative stress via regulating ΔΨm against neuronal insult in primary cortical neurons and brain mitochondria isolated from rat cortices. Results demonstrated that nobiletin (100 μM) treatment significantly increased cell viability against glutamate toxicity (100 μM, 20 min) in primary cortical neurons. Real-time imaging based fluorometry data proved that nobiletin evoked partial mitochondrial depolarization in these neurons. Nobiletininduced partial mitochondrial depolarization in intact neurons was also confirmed in the isolated mitochondrial model using a fluorescence microplate reader. Nobiletin significantly reduced mitochondrial calcium overload and reactive oxygen species (ROS) generation in glutamate (100 μM)-stimulated cortical neurons. In the isolated mitochondrial model, nobiletin decreased ROS production when treated with high concentrations of Ca2+ (5 μM) to mimic glutamate toxicity. In experiments to determine whether nobiletin has its own ROS scavenging ability, CPE and hesperidin exhibited considerable ROS scavenging activity, but nobiletin did not exhibit such an effect. Furthermore, nobiletin effects on basal ΔΨm were completely abolished in isolated brain mitochondria maintained in K+-free medium. To elucidate the molecular target of nobiletin, iberiotoxin 10 nM and 5-Hydroxydecanoate (5-HD) 500 μM were used to inhibit mitochondrial large-conductance Ca2+-activated K+ channels (mitoBKCa) and mitochondrial ATP-sensitive K+ channels (mitoKATP), respectively. The results suggested that K+ influx into the mitochondrial matrix by nobiletin is likely mediated by mitoBKCa and mitoKATP. In addition, nobiletin markedly reduced rotenone-induced mitochondrial ROS generation, suggesting the possibility that complex I of ETC might be another mitochondrial target of nobiletin. However, more detailed studies should be conducted to determine the exact molecular targets of nobiletin in mitochondria. Taken together, results demonstrate that nobiletin-induced partial depolarization of ΔΨm decreased mitochondrial Ca2+ overload and ROS generation, which are the important parameters of neuronal cell death. The K+ influx into the mitochondria matrix is critically engaged in the partial mitochondrial depolarization-related neuroprotective effect of nobiletin. Nobiletin-induced mitochondrial K+ influx is likely mediated, at least in part, by the activation of mitochondrial K+ channels.